Through studies of the inherited immunodeficiency X-linked lymphoproliferative disease (XLP), we discovered that the adaptor molecule SAP is required for development of natural killer T (NKT) cells, a subset of regulatory lymphocytes with essential functions in innate and adaptive immunity. In particular, we showed that XLP is caused by mutations in the SAP/SH2D1A gene and that SAP-deficient humans and mice lack NKT cells. To extend these findings and exploit them for more general therapeutic purposes, we plan to characterize the signaling mechanisms used by SAP to regulate NKT cell ontogeny and function. In T lymphocytes, SAP recruits the Src family tyrosine kinase Fyn to the cytoplasmic domain of the SLAM receptor, thereby promoting downstream phosphotyrosine signals that are required for TH2-type cytokine production. Our preliminary studies suggest that SAP controls NKT cell ontogeny through Fyn, but also through novel Fyn-independent pathways. Based on these observations, we hypothesize that SAP is a critical molecule required for NKT cell development. Furthermore, SAP may promote NKT cell ontogeny via signaling pathways distinct from those used by this adaptor in other cell lineages, such as CD4+ T cells. Because NKT cells have potent immunoregulatory properties, we also propose that the NKT cell defect contributes to the phenotypes of SAP-deficiency, including enhanced susceptibility to infection, humoral insufficiency and development of lymphoma. We will investigate these hypotheses through 3 specific aims. In Aim 1, we will use in vitro culture methods and in vivo mouse studies to explore the biochemical pathways involving SAP that guide NKT cell ontogeny. In Aim 2, we will manipulate SAP expression and function in mature NKT cells to determine how SAP-dependent signals control NKT cell functions, such as cytokine production and cytotoxicity. In Aim 3, we will use adoptive cell transfer to restore NKT cell function in NKT cell-deficient and Sap-/- mice to determine the importance of this lineage during clearance of virus infection and regulation of anti-viral immunity. The medical relevance of NKT cells is established, but the molecular mechanisms controlling their development and function are not well understood. Our analyses of XLP identify SAP as a crucial regulator of NKT cell ontogeny and illustrate how studies of a rare disorder can produce important insights into human biology and disease pathogenesis. By further defining the role of SAP in NKT cells, we hope to identify novel signaling pathways that can be targeted to improve the treatment of XLP as well as more common human diseases, such as immunodeficiency, autoimmunity and cancer.